Lung cancer is the leading cause of cancer mortality worldwide, with non-small cell lung cancer (NSCLC) the predominant histologic subtype of lung cancer and lung adenocarcinoma the major subset of NSCLC. Despite recent clinical progress with the use of specific targeted therapies, drug resistance remains a problem that limits patient survival. A promising strategy to combat cancer drug resistance is to deploy rational upfront polytherapies that suppress the survival and emergence of resistant tumor cells. However, in most tumors with oncogenic receptor kinases, the optimal initial polytherapy strategy is unclear because receptor kinases typically engage multiple effector pathways, and which of these individual pathways, if any, is most critical to tumor cell survival is poorly defined. We recently demonstrated in models of NSCLC harboring the recurrent oncogenic ALK receptor kinase fusion (EML4-ALK or ALK+) that the RAS-MAPK pathway, but not other known ALK effectors, is required for tumor cell survival. We revealed that EML4-ALK drives RAS-MAPK signaling by engaging all three major RAS isoforms (H, N-, K-RAS) via the HELP domain of EML4. MAPK pathway reactivation via either genomic amplification of KRASWT (wild-type) or downregulation of the MAPK phosphatase DUSP6 promoted resistance to ALK inhibition. Accordingly, upfront ALK and MEK co-inhibition enhanced both the magnitude and duration of initial response in EML4-ALK NSCLC in vitro and in vivo models. Furthermore, genomic amplification (or gene duplication) of KRASWT or downregulation of DUSP6 was observed in ALK+ lung adenocarcinoma patients with acquired ALK inhibitor resistance. Together, our findings provided new insight into the function of RAS-MAPK signaling in EML4-ALK NSCLC and the rationale for upfront ALK + MEK inhibitor co-treatment to improve patient outcomes, a novel clinical trial we are leading. Moreover, the findings indicated an unanticipated role of the EML4 partner in EML4-ALK oncogene function and RAS signaling. Here, we will further extend our initial discovery to test the overall hypothesis that RAS activation and signaling is a hallmark of oncogenic ALK function in NSCLC. In Aim 1, we will define the biological basis of RAS-MAPK signaling and dependence in EML4-ALK NSCLC, dissecting the molecular and cell biological control mechanisms governing RAS activation and signaling in ALK+ tumors. In Aim 2, we will define the mechanism(s) that may limit curative response to ALK + MEK inhibitor polytherapy in ALK+ NSCLC patients, levering cutting-edge CRISPR-based genetic screening studies and patient tumor samples from our ALK + MEK inhibitor clinical trial. Overall, these multi-disciplinary, collaborative, patient-focused studies spanning biochemical, genetic, pharmacologic, cell biological, and patient cohort and tumor molecular analysis will provide fundamental insight into the function and control of RAS and oncogenic ALK signaling in cancer and further enhance our novel rational polytherapy strategy. Our ultimate goal is to ensure we transform ALK+ NSCLC from a lethal disease into a chronic or curable condition through biologically-based precision medicine.